Molecular Biology Protocols

 
Marine DNA Sequencing and Analysis Center
Mount Desert Island Biological Laboratory


Last update June 23, 2005. 
Send comments to dtowle@mdibl.org

 

3A.  Purification of Total RNA from Animal Tissues: Phenol-Based Procedure

 

PREPARATION FOR RNA PURIFICATION

One of the most difficult steps in molecular cloning by RT-PCR is preparation of intact RNA.  Because degrading RNAses are very common – in every tissue and on every surface – much care must be taken to inactivate them and avoid contamination.  We will use disposable plasticware in every step possible and will wear gloves to reduce the likelihood of introducing RNAse from our own bodies.  Frequent changing of gloves will reduce the possibilities of contamination.

 

Treatment of Homogenizer Probe with RNAseZap (Ambion)

Following an RNA preparation, disassemble and clean the homogenizer probe.  Treat with RNAseZap by spraying all parts over a glass dish.  Rinse with Nanopure water then with nuclease-free water and allow to dry.  Treat the probe tool with RNAseZap as well.  After drying, reassemble probe (wearing fresh gloves) and wrap with aluminum foil for the next use.  Treat dissecting instruments in the same way. 

 

Solutions

Allow the Phenol:Chloroform:Isoamyl Alcohol mixture to stand at room temperature for 15 minutes or so to separate the phases.  The desired material is the lower organic phase.

 

Animals

Place crabs or other ectotherms in ice 20-30 minutes prior to dissection.  Treat other sources according to appropriate animal care and use guidelines.

 

Centrifugation

For homogenizing and centrifuging, use sterile disposable centrifuge tubes, not the reusable tubes recommended in the Promega protocol.  We will centrifuge 50-ml or 12-ml disposable tubes in a swinging bucket rotor at approximately 5,000xg for twice the time recommended in the Promega protocol.  Two-ml microcentrifuge tubes will be spun at the recommended g forces.  Cool the centrifuge and rotor to 4C.

 

RNA PURIFICATION PROCEDURE:  PROMEGA TOTAL RNA KIT

 

Day 1

 

Wear gloves throughout this procedure.  Change gloves frequently.  Do not contaminate gloves with face oils or doorknob RNAse!

 

1.  The procedure may be scaled down for preparing total RNA from small amounts of tissue (see the Promega protocol for details).  For this experiment, we will use 0.25 gm (or less) and 3 ml of denaturing solution. Add 3 ml of denaturing solution to a 12-ml sterile disposable polypropylene centrifuge tube and chill on ice for at least 5 minutes.  Weigh and record the weight of each tube with denaturing solution.

 

2.  Remove tissue with RNAseZapped instruments and (optional) rinse briefly in cold sterile phosphate-buffered saline (PBS). 

 

3.  Place the tissue directly into the 3 ml of denaturing solution and reweigh the tube.  Be sure that no more than 0.25 gm are added. 

 

4.  Homogenize the tissue in the 12-ml centrifuge tube, 20 seconds or so, at high speed.  Rinse homogenizer probe in Nanopure water (at least 3 changes) then denaturing solution between tissues.  The denaturing solution may be reused for rinsing purposes.

 

5.  Add 0.3 ml 2M sodium acetate (pH 4) and mix thoroughly by inversion.

 

6.  Add 3 ml phenol:chloroform:isoamyl alcohol (lower organic phase).  Caution!  Perform this addition in the safety hood if possible.  Mix by inversion and shake vigorously for 10 seconds.  Chill on ice for 15 minutes.

 

7.  Centrifuge in the refrigerated table top centrifuge at 4,000 rpm for 50 minutes at 4C.

 

8.  Using the P-1000 pipet and a sterile 1000-μl tip with filter, carefully remove 2 ml (2 x 1000 μl) of the top aqueous phase (containing the RNA) and transfer it to a fresh 12-ml centrifuge tube.  Be VERY careful to avoid taking material from the interface (= DNA and protein).  Perform this step in the safety hood!

 

9.  Add an equal volume of isopropanol, mix thoroughly, and incubate the sample at -20C for 30 min, or (better) overnight.

 

10.  Dispose of the contaminated tips and tubes and liquid phenol waste in closed containers for later shipment to a hazardous waste facility.  Caution!!

 

Day 2

 

1.  Pellet the RNA by centrifugation at 4,000 rpm for 40 minutes at 4C.  Remove the supernatant by carefully pipetting, making sure that you do not remove the pellet containing the RNA. 

 

2.  Respuspend the RNA pellet in 2.5 ml of denaturing solution and vortex until the RNA is dissolved.  In some instances, heating to 65C may be required to resuspend the pellet.  Heating should be done for as short a time as possible.

 

3.  Add 2.5 ml isopropanol, mix thoroughly, and precipitate the RNA by incubating at -20C for at least 30 min, or (better) 2 hours.

 

4.  Pellet the RNA by centrifugation at 4,000 rpm for 40 minutes at 4C.  Remove the supernatant by careful pipetting, making sure that you do not remove the pellet, which contains the RNA.  

 

5.  Add 5 ml ice-cold 75% ethanol to the pellet, vortex to break up the pellet, and then centrifuge at 4,000 rpm for 40 minutes at 4C.  Remove the supernatant by careful pipetting.  Allow the ethanol to evaporate from the pellet by placing the open tubes on their sides in an RNAse-free environment.  Alternatively, cover tubes with parafilm, poke a hole in the parafilm with a pipet tip, and dry the pellet in a vacuum desiccator for 15-20 minutes.  Do not overdry.

 

6.  Resuspend the RNA in 100 μl (or less) of Promega RNAse-free water by vortexing, transfer to a nuclease-free screw-capped 2.0-ml microcentrifuge tube, and store at -20C in a non-frost-free freezer or for long term storage at –80C.

 

QUANTITATING RNA AND DNA BY UV SPECTROPHOTOMETRY

Nucleic acids absorb ultraviolet light strongly at a wavelength of 260 nanometers. Empirical data indicate that RNA and DNA concentrations measured in a 10-mm-light-path quartz cuvet may be estimated by the following relationships:

If A260 = 1, then the DNA concentration is approximately 50 µg/ml.

If A260 = 1, then the RNA concentration is approximately 40 µg/ml.

To make calculations simple, we dilute RNA solutions 1:250 (2 µl plus 498 µl of nanopure water), measure A260, and multiply that value by 10 to calculate RNA concentration in µg/µl.

Following are the calculations used to arrive at such a method:

A260 * 40 = µg RNA/ml

A260 * 40 / 1000 = µg RNA/µl

For a solution diluted 250-fold, we must multiply the left side by 250:

A260 * 40 * 250 / 1000 = A260 * 10 = µg RNA/µl

For DNA solutions, we dilute 1:200 (2.5 µl plus 497.5 µl nanopure water), measure A260, and multiply that value by 10 to calculate DNA concentration in µg/µl.

To check for protein contamination, we can measure absorbance of our nucleic acid solutions at 280 nm. A pure RNA preparation gives an A260/A280 ratio of 1.9-2.0, while a pure DNA preparation gives a ratio of 1.8 to 1.9. Phenol and other organic substances absorb UV at 230 nm. Particulate material is detected by light scattering at 325 nm.